The overall goal of this project is to examine the role of Cullin-5 (Cul5) in breast tumorigenesis. The Cul5 gene is a putative tumor suppressor since it is located on a region of chromosome 11 (q22-23) that is associated with loss of heterozygosity in breast cancer. Previously, the Principal Investigator's laboratory demonstrated that with 50 cases of matched breast tissue there is an approximately 2.2-fold significant decrease (paired t- test, P < 0.0001) in the expression of Cul5 mRNA in breast tumor tissue versus matched normal tissue. Also, three metastatic breast tumor samples demonstrated a trend for decreased expression (about 4.3-fold) of Cul5 mRNA versus matched normal tissue. This decrease in the expression of Cul5 in breast tumor tissue and metastatic breast tissue versus matched normal tissue supports the hypothesis that decreased expression of Cul5 may play a role in tumorigenesis by promoting proliferation and cell invasion and metastasis. To examine the role of decreased Cul5 expression in breast tumorigenesis, the following three specific aims are proposed. In Specific Aim 1, an RNA interference (RNAi) -mediated in vitro model of Cul5 knock-down will be optimized using siRNAs, a breast epithelial cell line (MCF-10A) and breast cancer cell lines (MCF-7, MDA-MB-231). In Specific Aim 2, the hypothesis that decreased expression of Cul5 promotes proliferation in breast-derived cell lines will be tested by performing cell counts, WST-1 proliferation assays and by measuring the incorporation of bromodeoxyuridine into DNA in Cul5 knock-downs versus cells transfected with a negative control siRNA. Also, the effect of Cul5 knock-down to modulate the expression of cell cycle-related genes will be evaluated using a human cell cycle and apoptosis cDNA array. In Specific Aim 3, the hypothesis that decreased expression of Cul5 promotes cell invasion and metastasis will be tested by examining the cell invasion of Cul5 knock-downs versus a negative control siRNA by performing in vitro chemokinesis and chemotaxis assays using Matrigel invasion chambers. Also, the effect of Cul5 knock- down to modulate the expression of metastasis-related genes will be evaluated using a human metastasis cDNA array. The proposed studies are expected to provide insight into the role of the putative tumor suppressor Cul5 in the development and metastatic spread of breast cancer. A better understanding of cancer-related genes, such as Cul5, is expected to lead to better therapies for patients. [unreadable] [unreadable] [unreadable] [unreadable]